Light collection from objects within a fluid column

ABSTRACT

An optical arrangement receives output light emanating from an object disposed within a fluid column that crosses an optical refraction boundary of the fluid column between the object and the optical arrangement. The optical arrangement modifies the output light such that the modified output light has an intensity that is more uniform than the output light.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/133,723 filed Dec. 24, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/133,531 filed Sep. 17, 2018 now U.S. Pat. No.10,908,065, issued Feb. 2, 2021. The entire disclosure of which isincorporated herein by reference.

BACKGROUND

Object discrimination techniques distinguish between objects ofdifferent types. These techniques are particularly useful to sortbiological cells according to cell type. Some cell sorting approachesrely on light emanating from the cells to determine their type. In someimplementations, cells traveling in a column of fluid are exposed to anexcitation light and light emanating from the cells in response to theexcitation light is detected. Cells of a first type produce output lightthat is different in some characteristic, e.g., wavelength and/orintensity, from cells of a second type. The differences in output lightemanating from the cells can be the basis for cell type discriminationand sorting.

SUMMARY

Some embodiments are directed to an optical arrangement configured toreceive output light emanating from an object disposed within a fluidcolumn. The output light crosses an optical refraction boundary of thefluid column between the object and the optical arrangement. The opticalarrangement modifies the output light such that the modified outputlight has an intensity that is more uniform than an intensity of theoutput light. For example, within a cross section of the fluid column,the intensity of the modified output light can be substantially uniformirrespective of a position of the object.

According to some embodiments, an optical apparatus includes the opticalarrangement and further includes a detector that detects the modifiedoutput light and provides an electrical signal responsive to themodified output light.

In accordance with some embodiments, a discrimination system includes anexcitation light source configured to generate excitation light and todirect the excitation light toward an object in a fluid column. Theobject emanates output light in response to the excitation light. Thesystem comprises an optical arrangement configured to receive the outputlight. The output light crosses an optical refraction boundary of thefluid column between the object and the optical arrangement. The opticalarrangement modifies the output light such that the modified outputlight has an intensity that is more uniform than an intensity of theoutput light, e.g., the intensity of the modified output light issubstantially uniform irrespective of a position of the object in across section of the fluid column. An optical detector is configured todetect the modified output light and to provide an electrical signalresponsive to the modified output light. Object type discriminationcircuitry discriminates between a first type of object and a second typeof object based on the electrical signal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a diagram of a system incorporating an optical apparatus inaccordance with some embodiments;

FIG. 1B shows an xy plane cross section of the fluid column in themeasurement region of the system of FIG. 1A;

FIG. 2A shows light emanating from an object located near the center ofthe fluid column with substantially no refraction of light at thefluid-air interface of the fluid column.

FIG. 2B shows light emanating from an object located in an upper portionof the elliptical core of the fluid column exhibiting refraction oflight at the fluid-air interface;

FIG. 3 illustrates development of an analytical formula for angulardependence of the in-plane light ray density as a function of positionx;

FIG. 4A provides a family of graphs showing the angular dependence ofradiance for different object positions;

FIG. 4B provides a family of graphs of the relative intensity of lightcollected from the fluid column with respect to object position alongthe x axis for different numerical apertures of the collection optics.

FIG. 5A provides a family of graphs of the angular dependence ofradiance for different positions of the object and showing regions ofexclusion;

FIG. 5B shows the relative intensity of light collected from the fluidcolumn with respect to object position along the x axis when no anglesare excluded, when rays having angles between −0.3 rad and +0.3 rad areexcluded, and when rays having angles between −0.4 rad and +0.4 rad areexcluded;

FIG. 6 is a flow diagram of an approach for identifying objectstraveling in a fluid column with reduced positional variation ofdetected output light in accordance with some embodiments;

FIG. 7 is a top view of a ray tracing simulation of an optical systemthat includes an optical apparatus in accordance with some embodiments;

FIG. 8 is a photograph of a split objective lens configured to reducethe positional variation of detected output light for objects in a fluidcolumn in accordance with some embodiments;

FIGS. 9A and 9B illustrate the simulated performance of the opticalapparatus of FIG. 7 ;

FIG. 10 is a top view of a ray tracing simulation of an optical systemthat includes an optical apparatus in accordance with some embodiments;

FIG. 11 illustrates an optical apparatus comprising an elongated maskfeature in accordance with some embodiments;

FIG. 12 depicts an optical apparatus comprising an elongated maskfeature that can be used in conjunction with a plate having an apertureaccording to some embodiments;

FIG. 13 illustrates an optical apparatus comprising a plate andelongated mask feature wherein the optical transparency of the platevaries smoothly with position in accordance with some embodiments;

FIG. 14 illustrates an optical apparatus comprising a plate andelongated mask feature wherein the optical transparency of the platevaries with position in accordance with some embodiments;

FIG. 15 is a diagram of an optical apparatus comprising plate and anelongated mask feature extending across an aperture formed as a unitarystructure in accordance with some embodiments;

FIGS. 16 through 18 illustrate various configurations of longitudinaledges of elongated mask features in accordance with several embodiments;

FIG. 19 is a photograph of an optical apparatus that includes a wiremask in accordance with some embodiments; and

FIG. 20 is a photograph of an optical apparatus that includes a platehaving an elongated mask feature extending across an aperture formed asa unitary structure in accordance with some embodiments.

The figures are not necessarily to scale. Like numbers used in thefigures refer to like components. However, it will be understood thatthe use of a number to refer to a component in a given figure is notintended to limit the component in another figure labeled with the samenumber.

DESCRIPTION

Embodiments described herein relate to devices, systems and methods fordiscriminating between different types of objects. The objects emanateoutput light in response to an excitation light that is directed towardthe objects in a fluid column, such as a flow stream. In someimplementations, cell types are distinguished based on the intensity ofthe output light emanating from the objects. Specific embodimentsdiscussed herein are directed to distinguishing between X chromosomesperm cells and Y chromosome sperm cells. It will be appreciated thatthe approaches of this disclosure can be applied more generally todistinguishing between any objects of different types so long as theoutput light emanating from one object type has a discernable differencein at least one characteristic when compared to the light emanating fromanother object type. In some examples provided, the fluid column is aflow stream that has a curved boundary or interface where refraction oflight may occur. For example, the curved boundary of the fluid columnmay be generally circular in cross section. The fluid column can bebounded by solid walls or may be jetted into the air. The objects maymove along the fluid column which may include a central core shaped by asheath fluid that at least partially surrounds the central core. Lightemanating from the objects encounters at least one optical refractionboundary between the objects and other materials, such as at theinterface between the fluid column and air.

Due at least in part to the refraction at the fluid-air interface, thelight collection efficiency external to the fluid column of lightemanating from objects within the column depends upon the position ofthe objects for systems in the prior art. Light collection efficiencythat varies with position is detrimental in applications where the lightemanating from the objects must be precisely quantified and suchprecision is limited by random (not directly observable) positionfluctuations of the objects. The approaches disclosed herein enhance theprecision of systems that may be limited by such fluctuations, such asjet-in-air flow cytometers. As discussed in more detail below, thepositional variability of light intensity collected from objects in afluid column can be addressed by selectively masking rays at one or moreplanes (e.g, aperture stop, field stop) of the optical system in orderto reduce the dependence of intensity on position.

The approaches outlined herein are particularly applicable to flowcytometry. However, the approaches can be applied to any system wherelight is collected on one side of an interface from objects emanatingthe light from the other side of the interface, wherein the interfacecauses a variation in the emanating light ray paths in a mannerdependent on the object's position relative to the detector. Approachesherein modify the light collection efficiency of the output lightemanating from the object to compensate for positional variation withinthe fluid column.

The “jet-in-air” flow cytometer system 100 illustrated schematically inFIG. 1A is one type of flow cytometer that can be used to discuss theconcepts of the disclosure. The “jet-in-air” flow cytometer system 100pumps fluid into a chamber 110 at high pressures causing a flow stream150 comprising a fluid column to jet out of the exit nozzle 160 of thechamber 110 at high velocity, e.g., about 20 m/s. The fluid column 150expelled from the exit nozzle 160 can be roughly circular incross-section and may have a diameter of about 10 μm to about 100 μm insome implementations. The flow stream 150 is composed of a core stream151 within a sheath stream 152 where the arrows in FIG. 1A indicate thedirection of flow of the core and sheath streams 151, 152.

Within the chamber 110, a sample output nozzle 111 ejects the corestream 151 containing objects 171, 172 which may be of multiple types.The core stream 151 is bounded and shaped by a stream 152 of sheathfluid which is ejected from a sheath fluid nozzle (not shown) into thechamber 110. The sheath stream 152 at least partially surrounds the corestream 151, and the sheath stream 152 and the core stream 151 do notsubstantially mix. The sloping or angled walls 115 of the chamber 110cause the sheath stream 152 to narrow and/or maintain thecross-sectional size of the core stream 151 within the flow stream 150before and after the flow stream 150 is ejected from the exit nozzle 160of the chamber 110. The movement of the sheath stream 152 constrains theobjects 171, 172 in the core stream 151 to move toward the center of theflow stream 150 when the fluid column 150 is ejected from the chamber110. The flow stream 150 delivers the objects 171, 172 to a measurementregion 175 of the flow stream 150, e.g., in single file.

As the objects pass through the measurement region 175 of the flowstream 150, light from an excitation light source 180 providesexcitation light to the objects 171, 172. The excitation light source180 can provide light in a broad wavelength band or in a narrowwavelength band. For example, the excitation light source 180 may be alaser. In some configurations, the excitation light may be modified byan optical element 181. For example, the excitation light may be focusedon the measurement region 175 by a lens 181. Objects in the measurementregion 175 emanate light, e.g., scattered or fluorescent light, inresponse to the excitation light source 180.

Objects of a first type 171 will emanate light that differs in at leastone characteristic in comparison to light that emanates from objects ofthe second type 172. For example, in some scenarios, objects of thefirst type 171 will emanate light having a higher intensity than thelight that emanates from objects of the second type 172.

An optical collection arrangement 190 is arranged to collect the outputlight 161 emanating from the object within the measurement region 175that crosses the optical refraction boundary of the flow stream 150 atthe fluid-air interface. The optical arrangement 190 is configured tomodify the output light 161 to provide modified output light 162 thatcompensates for position dependence of the light emanating from theobject 172 a in the measurement region 175 as discussed in more detailbelow. A detector 185 receives the modified output light 162 and, inresponse, generates an electrical signal. In some scenarios, theamplitude of the electrical signal may be different for different objecttypes. The electrical signal is used by discrimination circuitry 187 todistinguish between different types of objects 171, 172. For example,the discrimination circuitry 187 may be configured to compare theamplitude of the electrical signal to a threshold to discriminatebetween objects of the first type 171 and objects of the second type172.

FIG. 1B shows an xy plane cross section of the flow stream 150 in themeasurement region 175. In the xy cross section of the measurementregion 175, the core stream 151 is elliptical in shape, and the fluid ofthe core stream 151 comprises at least one object 172 a suspended in abuffer solution. The sheath stream 152 is substantially surrounds thecore stream 151. In a particular example used for this discussion inthis disclosure, the objects 171, 172 are sperm cells and the system 100is implemented to discriminate X chromosome sperm from Y chromosomesperm.

A focused laser beam generated by the excitation source 180 illuminatesthe sperm cell 172 a within the measurement region 175. The cells 171,172 are stained with a fluorescent dye, and the excitation light causesthe cell 172 a within the measurement region to emanate fluorescentoutput light. The purpose of the elliptical core 151 is to orient asperm cell 172 a such that the flat sides of the sperm cell are facingto the left and the right as shown in FIG. 1B. In this orientation, theflat sides of the sperm cell 172 a face the laser 180 and the opticalcollection arrangement 190, respectively.

When the core stream 151 is elliptical, a sperm cell 172 a can take anynumber of positions along the x-axis within the core stream 151. FIG. 1Bshows three possible positions for the sperm cell 172 a in theelliptical core 151. In the orientation shown in FIG. 1B, the firstpossible position for the sperm cell 172 a in the core stream 151 isapproximately at the center of the elliptical core 151 (on the opticalaxis 199 of the optical collection arrangement 190), a second possibleposition is at the top of the core stream 151 (above the optical axis199), and a third possible position is at the bottom of the core stream151 (below the optical axis 199). A position-dependent refraction of theoutput light rays emanating from the sperm cell 172 a occurs at thefluid-air interface 153 at the different positions within the corestream 151.

When the sperm cell 172 a is located at the first position and the flowstream 150 has a circular cross section as shown in FIG. 1B, thein-plane rays of light emanating from the sperm cell 172 a areapproximately normally incident on the fluid-air interface 153. Raysthat emanate from points of the sperm cell 172 a away from its center,or rays that emanate out of the plane of the figure, are not exactlynormally incident on the interface 153; these rays are not considered inthis simplified discussion, but one of ordinary skill in the art can seehow the discussion could be generalized to include them. Thus, norefraction of light occurs at the fluid-air interface 153.

The diagram of FIG. 2A shows the absence of light refraction of theoutput light 298 emanating from a sperm cell 172 a and crossing theinterface 153 when the sperm cell 172 a is at the 1^(st) position withinthe elliptical core 151 shown in FIG. 1B. Correspondingly, the in-planedensity of the light rays 298 exiting the flow stream 150 in FIG. 2A isuniform with respect to ray angle. Uniform angular density of light rayscorresponds to uniform radiance as a function of ray angle.

In contrast, when a sperm cell 172 a is off the optical axis 199 and isnearer to the top or bottom of the elliptical core 151, e.g., at the2^(nd) and 3^(rd) positions of the elliptical core 151 shown in FIG. 1B,at least some of the output rays emanating from the sperm cell 172 aencounter the fluid-air interface 153 at an oblique angle. These outputrays are refracted at the fluid-air interface 153 in contrast to thenormal incidence scenario discussed above. The most oblique rays are themost severely refracted. Refraction of the light rays causes theradiance distribution of the fluorescent light exiting the flow stream150 across the fluid-air interface 153 to become non-uniform and to varywith position of the cell 172 a along the x axis. That is, thisrefraction changes the radiance distribution of output light emanatingfrom sperm cell 172 a outside of the flow stream 150.

For example, when the cell 172 a is located off the optical axis 199,e.g., at the 2^(nd) or 3^(rd) positions shown in FIG. 1B, the density oflight rays and thus the radiance on the air side of the interface 153 ishigher at positive or negative ray angles, respectively, with respect tothe optical axis 199 when compared to the radiance on the air side ofthe interface 153 at angles parallel to the optical axis 199 or atnegative or positive ray angles, respectively. Positive and negativerefer to the sign of the ray angle γ in FIG. 3 . FIG. 2B is a diagramillustrating light rays 299 emanating from a cell 172 a and exiting theflow stream 150 through the fluid-air interface 153 when the cell 172 ais located at the 2^(nd) position of the elliptical core 151. In thisscenario, the density of light rays, or radiance, at positive ray anglesis greater than the density of the light rays parallel to optical axis199 or at negative ray angles. For an optical system with apredetermined numerical aperture (NA), the amount of light collected bythe system from cells of the same type (e.g., the collection efficiency)may vary depending on whether the cell is in the first position or thesecond position. The positional dependence of the system collectionefficiency leads to inaccuracies in determining cell type.

With reference to FIG. 3 , an analytical formula for the light raydensity as a function of ray angle γ and sperm position x is determinedusing Snell's law, where γ is the angle of a light ray, with respect tothe optical axis, emanating from the object after refraction at thefluid-air interface. This analysis considers only rays within, ortangential to, the two-dimensional cross-section of the flow stream.

We wish to solve for the density of the light rays with respect to theangle γ, which we can use to determine the density of rays at theentrance pupil of an optical collection system for each sperm positionx. This can be written:

I _(γ)(γ).  (1)

For our purposes we can assume that the sperm cell emanates lightuniformly in all directions, so the density of emanated light rays withrespect to the angle θ is:

I _(θ)(θ)=1/π,  (2)

that is, uniformly distributed from

${\theta = {- \frac{\pi}{2}}}{to}{\theta = {\frac{\pi}{2}.}}$

By geometrical analysis:

$\begin{matrix}{{\theta = {\tan^{- 1}\left( \frac{{cos\varnothing} - x}{sin\varnothing} \right)}};} & (3)\end{matrix}$∝=π/2−Ø−θ; and  (4)

γ=π/2−Ø−β  (5)

wherein the angles γ, θ, ϕ, α, β, and the distance x are shown in FIG. 3. As the flow stream has index of refraction n, Snell's law yieldsanother relation between the angles:

sin β=n sin ∝.  (6)

The density of light rays external to the interface I_(β)(β) is relatedto the density of light rays internal to the interface I_(α)(α) by thefollowing formula, with T(α) representing the average, across bothpolarizations, of the transmission through the interface:

$\begin{matrix}{{I_{\beta}(\beta)} = {{I_{\theta}( \propto )}{T( \propto )}{❘\frac{d \propto}{d\beta}❘}}} & (7)\end{matrix}$

The transmission is related to the Fresnel reflection coefficients fors- and p-polarization, R_(s)(α) and R_(p)(α), with the followingformulas:

$\begin{matrix}{{{T( \propto )} = {1 - {R( \propto )}}},} & (8)\end{matrix}$ $\begin{matrix}{{{R( \propto )} = \frac{{R_{s}( \propto )} + {R_{p}( \propto )}}{2}},} & (9)\end{matrix}$ $\begin{matrix}{{{R_{s}( \propto )} = {❘\frac{\cos \propto {- {ncos\beta}}}{\cos \propto {+ {ncos\beta}}}❘}^{2}},{and}} & (10)\end{matrix}$ $\begin{matrix}{{R_{p}( \propto )} = {{❘\frac{{{cos\beta} - {ncos}} \propto}{{{cos\beta} + {ncos}} \propto}❘}^{2}.}} & (11)\end{matrix}$

Using Eq. (7) with the above and the following additional relations:

$\begin{matrix}{{{I_{\varnothing}(\varnothing)} = {{I_{\theta}(\theta)}{❘\frac{d\theta}{d\varnothing}❘}}},} & (12)\end{matrix}$ $\begin{matrix}{{{I_{\propto}( \propto )} = {{I_{\varnothing}(\varnothing)}{❘\frac{d\varnothing}{d \propto}❘}}},{and}} & (13)\end{matrix}$ $\begin{matrix}{{{I_{\gamma}(\gamma)} = {{I_{\beta}(\beta)}{❘\frac{d\beta}{d\gamma}❘}}},} & (14)\end{matrix}$

we have an expression for the density of rays with respect to γ:

$\begin{matrix}{{I_{\gamma}(\gamma)} = {{I_{\theta}(\theta)}{❘\frac{d\theta}{d\gamma}❘}{T(\gamma)}}} & (15)\end{matrix}$

Now, the optical collection arrangement's NA is given by the sine of themaximum ray angle γ₀, so we can solve for this angle in terms of NA:

γ₀=sin⁻¹(NA)  (16)

Finally, the relative collected light intensity, as a function of spermposition x, is given by integrating Eq. (15) from −γ₀ to γ₀ andnormalizing by that integral value at x=0:

$\begin{matrix}{{{Relative}{Intensity}} = \frac{\int_{- \gamma_{0}}^{\gamma_{0}}{I_{\gamma}\,{d\gamma}}}{\int_{- \gamma_{0}}^{\gamma_{0}}{{I_{\gamma}\,\left( {x = 0} \right)}{d\gamma}}}} & (17)\end{matrix}$

Using the formula for ray density distribution of Eq. (15), the angulardependence of ray density (radiance) for different sperm positions canbe plotted as in FIG. 4A. In FIG. 4A, each of the lines represents thedensity of rays as a function of angle γ for a given sperm position x,where the angle γ is in radians. The plots correspond to a series ofpositions that lie in a range symmetric about x=0, (corresponding tograph 404 in FIG. 4A), which is where the ray density (radiance) isuniform as a function of angle. When x is positive (e.g., 2^(nd)position in FIG. 1B, corresponding to graph 402), relative radiance ishigher for positive ray angles γ and lower for negative ray angles γ,and the opposite is true when x is negative (e.g., 3^(rd) position inFIG. 1B, corresponding to graph 403).

If the numerical aperture of the collection optics (optical collectionarrangement 190 in FIGS. 1A and 1B) is large, e.g., approaching one, thevariation in collected optical intensity with respect to position forlight emanating from an object within the elliptical core is relativelysmall. This is because essentially all light emanating from the objectand directed to the right would be collected by the collection optics,regardless of the exact ray direction, and the total amount of emanatinglight is invariant to object position (given uniform excitation). Incontrast, a small numerical aperture results in a relatively largecollected intensity variation with respect to object position, becausechanges in object position affect the radiance distribution, and a smallnumerical aperture implies only a portion of this changing radiancedistribution is collected. Practical systems may have NAs that aresignificantly less than one, e.g., less than 0.5, or less than 0.3. Thefamily of graphs provided in FIG. 4B illustrates the relative intensityof light collected from an object, as a function of object position x,through collection optics with different NAs. FIG. 4A illustrates therange of angles γ captured by the different numerical apertures of FIG.4B.

In the family of graphs of FIG. 4B, graph 412 shows the relativeintensity with respect to position along the x axis for collectionoptics (e.g., optical collection arrangement 190 shown in FIGS. 1A and1B) having a numerical aperture (NA) of 0.2; graph 414 shows therelative intensity with respect to position along the x axis forcollection optics having an NA of 0.4; graph 416 shows the relativeintensity with respect to position along the x axis for collectionoptics having an NA of 0.6; graph 418 shows the relative intensity withrespect to position along the x axis for collection optics having an NAof 0.8; and graph 419 shows the relative intensity with respect toposition along the x axis for collection optics having an NA of 0.9. Itis clear from FIGS. 4A and 4B that collection optics having smaller NAsproduce a larger variation in collected light intensity with respect toobject position when compared to collection optics having larger NAs.Additionally, collection optics with larger NAs collect light rayshaving a wider range of refraction angles than collection optics havingsmaller NAs, and therefore have a higher overall collection efficiency.

Various embodiments disclosed herein are directed to collection devices(e.g., arrangement 190 shown in FIGS. 1A and 1B) that reduce thevariation in collected light intensity with respect to object positionin a flow stream. Some embodiments discussed herein can provide modifiedoutput light that has less than about a 3%, or less than about a 2%, oreven less than about a 1% measured intensity variation for a deviationin position of the object that is less than 60% of a radius of the flowstream away from a center of the flow stream along an axis perpendicularto the optical axis. Many applications are sensitive to intensitymeasurement errors, which may arise from a variety of sources. Due tothe difficulty in reducing intensity fluctuations by preciselycontrolling the position of objects within the flow stream, it is usefulto instead reduce the variation in collected light intensity withrespect to object position by careful design of the optical collectionarrangement. For applications such as X/Y sperm sorting, it is often thecase that two or more cell populations are to be separated based on thedifference in measured fluorescence intensity between the populations.If the random position fluctuations lead to fluctuations in collectedlight intensity that are greater in magnitude than the nominaldifference in fluorescence intensity of the two populations, it is notpossible to distinguish them with simultaneously high yield and highpurity. The fluorescence intensity difference between X and Y spermcells is typically only a few percent (e.g., ˜4% for bovine sperm).Current sperm sorter systems can in theory achieve high throughput byincreasing the flow rate of the core stream, but this has the effect ofincreasing the width of the core stream. Consequently, there would be alarge uncertainty of the sperm position within the core of the flowstream. This position uncertainty and the resultant fluctuations incollected fluorescence intensity limit the maximum throughput of currentsperm sorter systems to levels which do not obscure the smallfluorescence intensity difference between X and Y sperm.

In sperm sorter applications, the sperm cells may be stained withHoechst 33342 (Ho33342), a cell-permeable dye that enters the cellnuclei and binds selectively to A-T base pairs in the minor groove ofdouble-stranded DNA within the sperm head of live cells. Typically, a UVlaser is used to excite the stained sperm cells. When excited optically(at or near 350 nm), Ho33342 stained Y-chromosome bearing (male) andX-chromosome bearing (female) sperm can be resolved by measuring a smalldifference in total fluorescence from each cell. The difference in totalfluorescence is proportional to the amount of stain within the spermcell, which is proportional to the chromosomal content. This differencevaries between mammalian species, but in domestic animals it is on theorder of 4%.

One approach for intensity—position compensation is evident in FIGS. 4Aand 4B. The brackets in FIG. 4A highlight regions of integration thatcorrespond to fluorescence collection optics with a given NA. Graphs ofthe collected intensity variation with respect to object position forthe NAs of FIG. 4A are provided in FIG. 4B. In FIG. 4B, for a given NA,integration over the fluorescence collection region is performed suchthat the intensity of collected light can be plotted as a function ofeach sperm position. It is evident from FIG. 4B that increasing the NAof the collection optics helps to decrease the influence of objectposition on the fluorescence intensity gathered via the collectionoptics.

Embodiments described herein relate to collection optics (e.g., theoptical collection arrangement 190 in FIGS. 1A and 1B) that reducecollected light intensity variation with respect to object position asdescribed above. According to some embodiments, the collection opticsoperate by masking rays in “angle space”, that is, the collection opticsselectively collect, attenuate, and/or block rays from different anglesγ in order to achieve a desired intensity vs. position profile. Inpractice, an “angle space” masking function can be applied at a pupil(e.g., entrance pupil, exit pupil, or aperture stop) of an opticalsystem, where the position of a ray intersection with the pupil planecorresponds to the angle γ. In some embodiments, the collection opticalarrangement achieves a desired, e.g., flatter, intensity vs. positionprofile by preferentially collecting higher angle (pointing away fromthe optical axis) light rays over lower angle light rays.

FIGS. 5A and 5B illustrate how excluding low-angle refracted rays, at agiven NA, causes the intensity-vs-position curve to flatten out.Excluding the low angle rays excludes the rays that produce the mostvariation in the intensity vs. position profile, whereas the angularvariation of radiance at high positive angles tends to cancel thecorresponding variation at high negative angles. FIG. 5A shows plots ofthe relative radiance vs. ray angle, γ, for different positions of theobject along the x axis where the angle γ is in radians. In FIG. 5A,each graph corresponds to an object position, x, within the core of aflow stream, as indicated in FIG. 3 . The brackets in FIG. 5A show theportion of the light rays that will be excluded by the collection opticsfor each position x, when rays having angle magnitude less than 0.3 radare excluded (bottom bracket in FIG. 5A) and when rays having anglemagnitude less than 0.4 rad are excluded (top bracket in FIG. 5A).

FIG. 5B shows the relative collected light intensity vs. position of theobject along the x axis when no angles are excluded (graph 500), whenrays having angles between −0.3 rad and +0.3 rad are excluded (graph503) and when rays having angles between −0.4 rad and +0.4 rad areexcluded (graph 504). Graph 5B shows that when lower angle rays areexcluded, the relative intensity vs. position graph exhibits lessintensity variation with respect to position.

An approach for identifying objects traveling in a fluid column in thepresence of positional variation is illustrated in the flow diagram ofFIG. 6 . The process includes modifying 620 output light emanating fromthe object passing through a cross section of a flow stream such that anintensity of the modified output light is more uniform than theintensity of the unmodified output light. In some embodiments, themodified output light is substantially uniform irrespective of aposition of the object. The modified output light is detected 630 and anelectrical signal is generated 640 in response to the detected modifiedoutput light. A processor or other circuitry may use the electricalsignal to discriminate 650 between objects of different types. Forexample, the circuitry may compare the amplitude of the electricalsignal (which corresponds to the intensity of the detected light) to athreshold value to discriminate between objects of a first type andobjects of a second type. Optionally, in some implementations,excitation light may be generated 610 by an excitation source anddirected to the cross section of the flow stream wherein the objectemanates the output light in response to the excitation light.

FIG. 7 is a top view of a ray tracing simulation of an optical system700 that includes an optical apparatus 710 in accordance with someembodiments. Apparatus 710 effectively extends the NA of the collectionoptics in the plane of a cross section of a fluid column, preferentiallycollecting higher angle rays that are more balanced in terms of positionvs. intensity over lower-angle rays that tend to inject variation intothe position vs. intensity profile, as explained in the discussion ofFIGS. 5A and 5B. Optical collection arrangement 710 modifies lightemanating from an object in a cross section of a flow stream such thatan intensity of the modified output light is more uniform than theoutput light emanating from the object. The modified output light can besubstantially uniform irrespective of a position of the object withinthe cross section. In this particular embodiment, the intensity of themodified output light is substantially uniform irrespective to theposition of the object along an axis perpendicular to an optical axis ofthe collection arrangement. Other embodiments may cause an intensity ofthe modified output light to be substantially uniform independent of aposition of the object along another axis, such as the optical axis ofthe collection arrangement.

The optical collection arrangement 710 preferentially collects lightrays emanating from the object at higher angles with respect to theoptical axis 799 of the optical arrangement over light rays emanatingfrom the object at lower angles with respect to the optical axis 799. Insome implementations, the optical collection arrangement 710 is a splitobjective lens. A first section 711 of the split objective lens 710collects a first portion 751 of the higher angle light emanating fromthe object (object not shown in FIG. 7 ). A second section 712 of thesplit objective lens 710 collects a second portion 752 of the higherangle light emanating from the object. As shown in FIG. 7 , in someembodiments, a mask that further prevents the collection of lower anglerays may be disposed anywhere that the lower angle rays would beblocked, e.g., near an aperture stop or pupil plane, where the light iscollimated. For example, a mask 786 may be disposed between the twolenses 711, 712 as shown in FIG. 7 .

As indicated in FIG. 7 , the system 700 may be implemented as a foldedoptical system using mirrors 721, 722, 723, 724 to redirect thecollected portions of light along the optical axis 799 of the system 700and toward the detector 785. Mirrors 721, 722 redirect the first portion751 of light toward and along the optical axis and mirrors 723, 724redirect the second portion 752 of light toward and along the opticalaxis 799. As illustrated in FIG. 7 , the system 700 may optionallyinclude a filter 730 such as an optical bandpass or longpass filterconfigured to substantially attenuate the excitation light. The system700 can include lens 740 configured to focus the first and secondportions 751, 752 of the light toward the detector 785.

FIG. 8 is a photograph of a split objective lens configured to reducethe variation of intensity with respect to object position. It should benoted that the split-objective design allows the fluorescence collectionoptics to be placed closer to the flow stream than would otherwise bepossible, due to the spatial obstruction caused by the nozzle generatingthe flow stream. A single lens with the same effective NA as the splitobjective would be too big to place its focal point immediately belowthe nozzle generating the flow stream. The optical detection of objectswithin the flow stream is optimally performed immediately after the flowstream exits the nozzle, where the stream is most stable, therefore itis important to have high NA optics that do not interfere with thenozzle.

FIG. 9A illustrates the simulated performance of the split objectivelens based on the model in FIG. 7 vs. the simulated performance of thecomparative arrangement shown in FIG. 10 without the use of the spatialmask 1010. Graph 901 provides the intensity with respect to objectposition for a system that includes the split objective lens discussedabove. Graph 902 is the intensity vs. position of the comparativearrangement and is provided in FIG. 9A for comparison. Note the higheroverall collection efficiency of the split-objective arrangement inaddition to the decreased effect of position variation on collectedlight intensity.

In order to show this comparison more clearly, in FIG. 9B, each graph901 a, 902 a is normalized to 100%. Graph 901 a provides the collectionefficiency relative to center position for the split objective lensarrangement. Graph 902 a shows the collection efficiency relative tocenter position for the comparative system with only a single objectivelens. The split objective lens yields less than 1% deviation from thecenter collection efficiency at an object position of 20 μm, whereas thecomparative arrangement yields over 10% deviation from the centercollection efficiency at the same object position.

It is possible to decrease variation in the position vs. intensityprofile using other approaches as well, all of which are considerednovel aspects of the disclosure. For example, rather than a collectionarrangement that selects rays in angle space (e.g., close to a pupil ofthe optical system), as exemplified by the split objective lens, it ispossible to select rays in image or position space (e.g., close to animage plane of the optical system). One example of an optical collectionarrangement that selects rays in position space is a spatial mask nearan image plane of the optical system. In order to make such a maskeasier to align and more robust to alignment, it helps to increase theoptical system magnification, allowing one to use larger mask featuresizes.

FIG. 10 is a top view of a ray tracing simulation of an optical system1000 that includes an optical collection arrangement 1010, e.g., aspatial mask, that attenuates light rays emanating from the object nearthe center of a flow stream cross section while not attenuating lightrays emanating from the object at the top and bottom of the flow streamcross section. The term “attenuating” as used herein encompassespartially blocking or fully blocking the light rays. For example, anattenuated light ray may have an intensity reduction of 25% or 50% or75% or even 100% when compared to its original intensity, wherein 25%,50%, and 75% attenuation corresponds to a light ray that is partiallyblocked and 100% attenuation corresponds to a light ray that is fullyblocked. The system 1000 shown in FIG. 10 includes a single objectivelens 1070 that collimates light emanating from the object (object notshown in FIG. 10 ). System 1000 optionally includes a filter 1030, suchas a bandpass filter or a longpass filter configured to block excitationlight from reaching the detector 1085. A lens 1040 can be used to focuscollected light toward the sensitive region 1086 of the detector 1085. Aspatial mask 1010 attenuates or blocks light rays emanating from thecenter of the flow stream cross section 1050 from reaching the detector1085 while not attenuating or blocking light rays emanating from the topand bottom regions 1051, 1052 of the flow stream cross section 1050 fromreaching the detector 1085. In FIG. 10 , the top region 1051 of the flowstream cross section 1050 refers to the portion of the flow stream crosssection that is above the optical axis 1099 in FIG. 10 . The bottomregion 1052 of the flow stream cross section 1050 is below the opticalaxis 1099 in FIG. 10 . The mask 1010 may be opaque to the emanatinglight or may be semi-transparent. In some embodiments, the opticaloptical transparency of the mask 1010 may vary with position, e.g., suchthat an image of the center of the flow stream cross section is moreattenuated than an image of the top and/or bottom of the flow streamcross section.

A simple spatial mask that alleviates the variation in collectedintensity with object position is a thin wire (for example, having adiameter in a range of about 100 microns to 300 microns, e.g., about 200microns in diameter) in front of the optical detector 1085 and near animage of the flow stream, with the wire axis oriented parallel to theflow stream and nominally centered with respect to the optical axis1099. The effect of the wire can be varied by moving it in and out ofthe image plane 1087, which is where an image of the flow stream appears(a magnified image in the current embodiment).

A spatial mask that reduces the variation in collected intensity withobject position is illustrated in FIG. 11 . The spatial mask comprisesan elongated feature 1110, which may be implemented as a thin wirehaving a circular cross sectional area, a bar having a rectangular crosssectional area or other mask feature disposed at least partially acrossthe active area 1185 of the optical detector and near an image of theflow stream. The elongated mask feature 1110 can be implemented in manyways, including an extruded metal wire, an etched metal feature, humanor animal hair, a trace deposited on a glass slide, or an ink lineprinted on a transparent medium.

Generally, the length of the mask feature, L, is much greater than itswidth, W. The mask feature 1110 may be oriented such that the length ofthe mask feature 1110 runs parallel to the flow stream and the maskfeature 1110 is nominally centered with respect to the optical axis ofthe system (see FIG. 10 ). In some embodiments, the mask feature 1110may have a width of about 100 microns to about 300 microns e.g., about200 microns.

As illustrated in FIG. 12 , an elongated mask feature 1210 can be usedin conjunction with a plate 1220 having an aperture 1222 wherein theelongated mask feature 1210 is disposed at least partially across theaperture 1222 as illustrated in FIG. 12 . The plate 1220 can beparticularly useful for alignment of the system optics to achieve theoptimal intensity difference between objects of two types with slightlydifferent intensities.

In some embodiments, the plate may be made of a material that partiallyblocks (blocks more than 25% and less than 75% of the light),substantially blocks (blocks more than 75% of the light), or completelyblocks (blocks 100% of the light) the light emanating from the objectunder test from reaching the active area 1230 of the detector. Theaperture 1222 in the plate 1220 transmits substantially all of the lightemanating from the object to the active area 1230 of the detector. Theplate 1220 and aperture 1222 facilitates alignment of the system optics,allowing the operator to align the mask feature 1210 such that optimalcontrast is achieved between the lower light intensity emanating from afirst type of object and the slightly higher light intensity emanatingfrom a second type of object

FIG. 13 illustrates another embodiment in which the optical transparencyof the plate varies across its length and width. In this example, theplate 1320 is more optically transparent closer to the aperture 1322 andis less transmissive farther from the aperture. However, the oppositescenario is also possible, wherein the plate is less transmissive nearthe aperture and more transmissive farther from the aperture.

FIG. 14 illustrates another version of a plate 1420 that has a stepwiseoptical transparency gradient. The plate 1420 becomes more opticallytransmissive closer to the aperture 1422 in distinct steps in regions1421 a, 1421 b, 1421 c, 1421 d. Comparing the plate 1320 of FIG. 13 withthe plate 1420 of FIG. 14 , the optical transparency of plate 1320 makesa gradual transition from the outer edges of the plate 1320 having loweroptical transparency transitioning to a higher optical transparencynearer the aperture 1322 in the center of the plate 1320.

In some embodiments, the aperture plate and the elongated mask featureextending across the aperture are formed as a unitary structure asillustrated in FIG. 15 . FIG. 15 shows a plate 1520 including anelongated feature 1510 that bisects the aperture 1522. In someembodiments the unitary aperture plate 1520 can have a opticaltransparency gradient as previously discussed and illustrated withreference to FIGS. 13 and 14 . A unitary aperture plate as in FIG. 15can be formed, for example, by photoetching a metal plate.

The longitudinal edges 1510 a, 1510 b of the elongated mask feature 1510need not be parallel as they are shown in FIG. 15 . In some embodimentsthe alignment process may be enhanced by having an elongated maskfeature 1610, 1710 with non-parallel longitudinal edges 1610 a, 1610 b,1710 a, 1710 b as shown in FIGS. 16 and 17 . In some embodiments thelongitudinal edges 1810 a, 1810 b of the elongated mask feature 1810 maybe curved as illustrated in FIG. 18 .

FIG. 19 is a photograph showing an optical arrangement comprising aplate 1920 having an aperture 1922 in accordance with some embodiments.An elongated mask feature 1910 comprising a thin wire is positionedacross the aperture 1922 in front of the entrance to a photomultipliertube detector. The wire modifies the output light emanating from objectsin the flow stream by preferentially attenuating light emanating fromobjects at the center of the flow stream cross section as previouslydiscussed. The preferential attenuation of light provides a more uniformlight intensity vs. object position profile when compared to theunmodified output light.

FIG. 20 is photograph showing an optical arrangement in accordance withsome embodiments comprising a plate 2020 and elongated mask feature 2010extending across an aperture 2022 formed as one unitary structure. Forexample, the plate and elongated mask feature 2010 may be formed byphotoetching the (split) aperture 2022.

The foregoing description of various embodiments has been presented forthe purposes of illustration and description and not limitation. Theembodiments disclosed are not intended to be exhaustive or to limit thepossible implementations to the embodiments disclosed. Manymodifications and variations are possible in light of the aboveteaching.

1. A discrimination system comprising: a measurement region configuredto receive a flow stream comprising a core stream containing objects,the core stream being surrounded by a sheath stream that defines anoptical refraction boundary; an excitation source configured to generateexcitation light directed towards the objects in the measurement region,the objects emanating output light in response to the excitation light;an optical arrangement having an optical axis configured to receive andmodify the output light emanating from the object disposed within a flowstream, the optical arrangement further comprising a mask forattenuating excitation light emanating from objects near the center ofthe flow stream; an optical detector arranged to receive the modifiedoutput light, the optical detector configured to detect the modifiedoutput light and to provide an electrical signal responsive to themodified output light; and object type discrimination circuitryconfigured to discriminate between a first type of object and a secondtype of object based on the electrical signal.
 2. The discriminationsystem of claim 1, wherein the mask comprises a wire.
 3. Thediscrimination system of claim 1, wherein the mask is configured topreferentially collect a first portion of the output light emanatingfrom objects at larger angles with respect to the optical axis relativeto a second portion of the output light emanating from the objects atsmaller angles with respect to the optical axis.
 4. The discriminationsystem of claim 1, wherein the mask further comprises a plate thatincludes an aperture and an elongated mask feature disposed at leastpartially across the aperture, the elongated mask feature arranged suchthat a longitudinal axis of the elongated mask feature is substantiallyparallel to a direction of flow of the objects in the flow stream, theplate and the elongated mask feature being less transmissive to theoutput light than the aperture.
 5. The discrimination system of claim 4,wherein the plate and the elongated mask feature are one unitarystructure.
 6. The discrimination system of claim 4, wherein theelongated mask feature is attached to the plate.
 7. The discriminationsystem of claim 4, wherein optical transparency of the plate to theoutput light varies as a function of position across the plate.
 8. Thediscrimination system of claim 4, wherein opposing longitudinal edges ofthe elongated mask feature are substantially parallel to one another. 9.The discrimination system of claim 4, wherein opposing longitudinaledges of the elongated mask feature are not parallel to one another. 10.The discrimination system of claim 4, wherein opposing longitudinaledges of the elongated mask feature are tapered along a longitudinalaxis of the elongated mask feature.
 11. The discrimination system ofclaim 1, wherein the mask is configured to preferentially attenuateoutput light received from a first position in the flow stream comparedto output light received from a second position in the flow stream,wherein the second position is farther from the center of the flowstream than the first position along an axis perpendicular to theoptical axis of the optical arrangement.
 12. The discrimination systemof claim 1, wherein the mask comprises an elongated mask feature thatattenuates the output light received from the first position, theelongated mask feature disposed proximate to an active area of theoptical detector such that a longitudinal axis of the elongated maskfeature is substantially parallel to a direction of flow of the objectsin the flow stream.
 13. The discrimination system of claim 12, whereinthe elongated mask feature has a round cross sectional area.
 14. Thediscrimination system of claim 12, wherein the elongated mask featurehas a rectangular cross sectional area.
 15. The discrimination system ofclaim 12, wherein the elongated mask feature is a metal wire.
 16. Thediscrimination system of claim 12, wherein the elongated mask feature isa trace deposited on glass.
 17. The discrimination system of claim 12,wherein the elongated mask feature is a filament.
 18. The discriminationsystem of claim 12, wherein the elongated mask feature is an ink lineprinted on transparent paper.
 19. The discrimination system of claim 12,wherein the elongated mask feature is disposed at about a center of anactive area of the detector.